A variety of post-translational modifications including methylation, sulfation, phosphorylation, lipid addition and glycosylation are performed by higher eukaryotes. Many of the secreted proteins, membrane proteins and proteins targeted to vesicles or certain intracellular organelles are known to be glycosylated. Glycosylation, the covalent attachment of sugar moieties to specific amino acids, is one of the most common, yet important post-translational modifications for recombinant proteins. Protein glycosylation has multiple functions in the cell including its essential role in protein folding and quality control, molecular trafficking and sorting, and cell surface receptor interaction.
N-linked glycosylation involves addition of oligosaccharides to an asparagine residue found in certain recognition sequences in proteins (e.g., Asn-X-Ser/Thr). N-linked glycoproteins contain standard branched structures which are composed of mannose (Man), galactose, N-acetylglucosamine and neuramic acids. High-mannose oligosaccharides typically include two N-acetylglucosamines with multiple mannose residues (5 or more). Glycoproteins produced in mammalian cell culture may contain varied levels of these high mannose (HM or HMN) glycoforms such as Mannose5 (Man5), Mannose6 (Man6), Mannose7 (Man7), Mannose8 (Man8) and Mannose9 (Man9).
While the glycoforms of a recombinant glycoprotein expressed by Chinese hamster ovary (CHO) host cell are largely determined by intrinsic genetic factors, high mannose glycoform content can also be affected by cell culture conditions (Pacis, et al., (2011) Biotechnol Bioeng 108, 2348-2358).
Glycosylation can affect therapeutic efficacy of recombinant protein drugs. The influence of glycosylation on bioactivity, pharmacokinetics, immunogenicity, solubility and in vivo clearance of therapeutic glycoproteins have made monitoring and control of glycosylation a critical parameter for biopharmaceutical manufacturing. The high mannose glycoform content of therapeutic proteins is a critical quality attribute that has been found to affect pharmacokinetic properties of certain therapeutic antibodies (Goetze, et al., (2011) Glycobiology 21, 949-59; Yu, et al., (2012) MAbs 4, 475-87). Therefore, methods for controlling the high mannose glycoform content of therapeutic proteins would be beneficial.
There is a need in the pharmaceutical industry to manipulate and control the high mannose glycoform content of recombinant therapeutic glycoproteins and methods for doing such would be useful. The invention provides a method for manipulating the high mannose glycoform content of recombinant glycoproteins by regulating ornithine metabolism in the host cells.